Effects Of Giardia Doudenalis Cathepsin B On The Tight Junction Function And Antimicrobial Peptide Expression Of Intestinal Epithelial Cells

Giardia doudenalis is a protozoan parasite that parasites in the duodenum of mammals and is the main cause of water-borne diarrhea worldwide.It breaks open in the duodenum and jejunum,releasing excyzoites that quickly differentiate to trophozoites.The trophozoites adhere to the apical surface of intestinal epithelial cells(IECs)with the adhesive disc,which led to a succession of pathophysiological changes such as diarrhea,malabsorption and weight loss.Cysteine proteases(CPs)have been identified as major virulence factors in protozoan parasites,and play important roles in pathogenesis during life cycles.Increasing interest has recently been directed towards parasite-secreted CPs,and more specifically cathepsin B(CTSB),which has served as virulence factors and targets for therapeutic intervention.Protozoan parasite CPs activity is implicated in intestinal barrier dysfunction,mucus depletion and microbiota biofilm alteration during infection.In the process of Giardia infection,cathepsin B can cause acute symptoms,and may also cause complications after chronic infection,such as irritable bowel syndrome.Tight junctions are localized at the most apical region of the intercellular space and are the first barrier against pathogens.During the co-culture period of Giardia and intestinal epithelial cells,Giardia trophozoites can destroy the tight junctions of intestinal epithelial cells.Cathelicidin LL-37 are small cationic antimicrobial peptides of the mammalian innate immune system with broad activity against bacteria and protozoa.This study tried to screen Giardia cathepsin B that disrupted cell tight junctions and reduced the expression of LL-37 during interaction between Giardia trophozoites and host cells.In this study,we first established a mouse model infected by Giardia.Detection of transcript and expression levels of tight junction protein in mouse duodenum by(Real-time fluorescence quantification PCR,q PCR)and Western blot analysis.The results showed that Giardia infection led to a significant decrease in the transcription levels of the mouse duodenal tight junction protein Claudin-1,Claudin-2,Occludin,ZO-1,ZO-2 and JAM(P < 0.05),while only Claudin-1,Claudin-4,Occludin and ZO-1 protein expression levels were decreased.At the same time,the transcription level of the antimicrobial peptide CRAMP was also significantly reduced(P < 0.05).The results showed that the weight of mice infected with Giardia was reduced,and the infected mice occurred diarrhea and the intestinal segment was swollen.HE staining show that after Giardia infection,the villi of small intestine became shorter,atrophic or even broken,the arrangement of intestinal epithelial cells was irregular,and the integrity of intestinal epithelial cell layer was destroyed.Giardia infection inhibited the expression of tight junction proteins of intestine.During interaction between Giardia and IECs.Firstly,q PCR and Western blot technology were used to evaluated expressions of a variety of tight junction proteins in IECs treated with or without Giardia.The results of q PCR indicated that Giardia stimulation could significantly reduce the transcription levels of the six tight junction protein Claudin-1,Claudin-2,Occludin,ZO-1,ZO-2and JAM in Caco-2 cells(P < 0.05),but the transcription level of Claudin-4 was up-regulated(P <0.05).Western blot results confirmed that Giardia stimulation reduced the protein expression of Claudin-1,Claudin-4,Occludin and ZO-1.Using immunofluorescence technology,it was observed that the stimulation of Giardia caused localization changes of Claudin-1.The Claudin-1 protein in the control group cells was localized on the cell membrane.After 6 h of co-cultivation with Giardia,Claudin-1 protein is located in the cytoplasm.Secondly,q PCR results showed that stimulation by Giardia could cause a significant down-regulation of transcription level of the intracellular antimicrobial peptide LL-37(P < 0.05).Since vitamin D receptor(VDR)can up-regulate the expression of LL-37,we tested the transcription level of VDR in Giardia-treated or untreated IECs.It was found that the transcription level of VDR was also significantly reduced(P < 0.05).This indicated that Giardia stimulation decreased the transcription level of LL-37 through cause the downregulating VDR transcription level.In this study,Giardia was pre-incubated for 30 min with 1μg/m L of E-64 or 10μg/ml of CA074 Me,then they were used to treated IECs.The results of q PCR and Western blot showed that both inhibitors could significantly alleviate the down-regulation of tight junction protein transcription and expression levels in Caco-2 cells caused by Giardia stimulation.At the same time,q PCR results showed that the down-regulation of LL-37 and VDR transcription levels was also restored by above inhibitors.These results indicated that Giardia cathepsin B played an important role in destroying tight junctions and helping Giardia evade the host’s immune process.In order to further study the effects of cathepsin B on the expression and location of the tight junction proteins in IECs.This study selected Giardia cathepsin B(CTSB-3 and CTSB-4)genes,using bioinformatics analysis to predict its hydrophilicity and hydrophobicity,signal peptide,domain,secondary structure and tertiary structure.In this study,primers were designed based on the two cathepsin B gene sequences,and the target genes of CTSB-3 and CTSB-4 were successfully amplified using Giardia genome sequence as a template.The recombinant expressed plasmids p Cold-TF-CTSB-1～6 and p Cold-TF-CTSB-8 were constructed using p Cold-TF vector,and transformed into competent cells BL21.The soluble recombinant proteins r CTSB-1～r CTSB-6 and r CTSB-8 were successfully expressed.The recombinant proteins r CTSB-1～r CTSB-6 and r CTSB-8were used to stimulate Caco-2 cells to explore the damage effects of recombinant Giardia cathepsin B on tight junctions.Western blot results showed that the recombinant proteins r CTSB-2,r CTSB-3,r CTSB-4,and r CTSB-6 could reduce the expression of tight junction protein Claudin-1,Claudin-4,Occludin and ZO-1 in IECs.r CTSB-5 could reduce the expression of Claudin-1 and Claudin-4,but has no effect on the expression of Occludin and ZO-1.r CTSB-1 and r CTSB-8 have no such effects.At the same time,the effects of recombinant protein stimulation on the localization of tight junction protein were detected by immunofluorescence.Furthermore,immunofluorescence images showed reduced levels and re-localization of the tight junction proteins claudin-1,after 6 h of incubation with the recombinant protein r CTSB-2,r CTSB-3,r CTSB-4 and r CTSB-6.The location of Claudin-1 in Caco-2 cells is transferred from the cell membrane to the cytoplasm.However,r CTSB-1,r CTSB-5 and r CTSB-8 have no such effects.The results of q PCR showed that the transcription levels of LL-37 and VDR were significantly down-regulated in r CTSB-3 and r CTSB-4 stimulated cells(P < 0.05),and other recombinant proteins had no effects on the transcription of LL-37 and VDR.Overall,this study found that during Giardia infection,the tight junction protein Claudin-1,Claudin-4,Occludin may be caused by CTSB-2,CTSB-3,CTSB-4 and CTSB-6 in cathepsin B.The decreased expression of ZO-1 and Claudin-1 in the cell changes,thereby destroying the intestinal barrier,leading to symptoms such as diarrhea and abdominal pain.CTSB-3 and CTSB-4 could reduce the transcription level of antimicrobial peptides in cells,help Giardia evade the host’s active immunity and facilitate the colonization of trophozoites in the intestine.This study attempts to reveal the role of various proteins of the Giardia cathepsin B family in destroying the tight junction function of host cells and innate immunity,and provides a theory for the further study of cathepsin B as a Giardia virulence factor.The basis will also be of great significance to prevention and treatment of giardiasis.